Abstract
EmrE in Escherichia coli belongs to the small multidrug resistance (SMR) transporter family. It functions as a homo-dimer, but the orientation of the two monomers in the membrane (membrane topology) is under debate. We expressed various single-cysteine EmrE mutants in E. coli cells lacking a major efflux transporter. Efflux from cells expressing the P55C or T56C mutant was blocked by the external application of membrane-impermeable SH-reagents. This is difficult to explain by the parallel topology configuration, because Pro55 and Thr56 are considered to be located in the cytoplasm. From both the periplasm and the cytoplasm, biotin-PE-maleimide, a bulky membrane-impermeable SH-reagent, could access the cysteine residue at the 25th position in the presence of transport substrates and at the 108th position. These observations support the anti-parallel topology in the membrane.
MeSH terms
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Antiporters / chemistry*
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Antiporters / genetics
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Antiporters / metabolism
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Biological Transport
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Biotin / pharmacology
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Cell Membrane Permeability
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Cysteine / genetics
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Cytoplasm / chemistry
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Cytoplasm / metabolism*
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Dimerization
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / metabolism
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Maleimides / pharmacology
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Membrane Transport Proteins / chemistry
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Membrane Transport Proteins / genetics
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Membrane Transport Proteins / metabolism*
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Multidrug Resistance-Associated Proteins / chemistry*
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Multidrug Resistance-Associated Proteins / genetics
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Multidrug Resistance-Associated Proteins / metabolism
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Mutation
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Periplasm / chemistry
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Periplasm / metabolism*
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Polyethylene / pharmacology
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Proline / genetics
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Protein Structure, Secondary
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Threonine / genetics
Substances
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Antiporters
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Escherichia coli Proteins
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Maleimides
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Membrane Transport Proteins
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Multidrug Resistance-Associated Proteins
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EmrE protein, E coli
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maleimide
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Threonine
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Biotin
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Polyethylene
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Proline
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Cysteine